Download Ubiquitination and Auxin Signaling: A Degrading Story

The Plant Cell, S81–S95, Supplement 2002, www.plantcell.org © 2002 American Society of Plant Biologists
Ubiquitination and Auxin Signaling: A Degrading Story
Stefan Kepinski and Ottoline Leyser1
Department of Biology, University of York, Box 373, York, United Kingdom
INTRODUCTION
Signaling pathways are rarely straightforward, and auxin
signal transduction is no exception. The diverse events in
which auxin is involved tell of the daunting complexity behind the plant’s response to auxin. This one molecule can
cause changes in rates of cell division and cell elongation,
alter ion fluxes across membranes, cue changes in patterning and differentiation, and affect the expression of hundreds of genes (Davies, 1995; Berleth and Sachs, 2001).
Does auxin act through multiple pathways to these diverse
ends, or is there a single pathway that is dependent on the
spatial, temporal, and environmental context in which auxin
is received? Or is it both? Although the answers to these
and other broad questions of auxin biology remain unclear,
we are now in a position to connect at least some areas of
research on auxin signal transduction into a more cohesive
whole. Recent advances in our understanding of the apparent hub of auxin signaling, between perception and downstream auxin-induced gene expression, has developed from
the mutational and molecular analysis of three main groups
of proteins: the auxin/indoleacetic acids (Aux/IAAs), the auxin
response factors, and components of the ubiquitin-mediated
proteolytic pathway. It seems that regulated protein degradation is central to most aspects of the auxin response.
THE UBIQUITIN PATHWAY
Ubiquitin-mediated proteolysis has emerged as being as
fundamentally important as phosphorylation in terms of its
involvement in diverse cellular events. Target proteins are
condemned to degradation in the 26S proteasome by the
addition of a polyubiquitin chain in what is essentially a
three-step process (Figure 1) (Voges et al., 1999; Jackson et
al., 2000; reviewed by Pickart, 2001). First, ubiquitin is activated by the ATP-dependent formation of a thiolester bond
with a conserved Cys residue of a ubiquitin-activating en-
1 To
whom correspondence should be addressed. E-mail hmol1@
york.ac.uk; fax 44-1904-434312.
Article, publication date, and citation information can be found at
www.plantcell.org/cgi/doi/10.1105/tpc.010447.
zyme, E1. Activated ubiquitin then is passed to one of a
family of E2 ubiquitin-conjugating enzymes, from which it is
conjugated to a Lys residue of the target protein, often with
the assistance of an E3 enzyme, ubiquitin protein ligase.
This process is reiterated such that one or two additional
ubiquitins are polymerized upon the first ubiquitin molecule.
Target proteins marked with less than four ubiquitins are
poor substrates of the 26S proteasome and often escape
degradation. In some cases, an additional step ensures efficient proteolysis by extending the ubiquitin chain, a process
that requires a multiubiquitin chain assembly factor, or E4
(Koegl et al., 1999; Azevedo et al., 2001).
Of course, ubiquitination must be directed against specific targets, and it is the E3 enzyme that mediates the critical step of substrate recognition, a fact reflected in the
hundreds of E3s of various classes that are encoded by the
eukaryotic genomes sequenced to date (Deshaies, 1999;
Voges et al., 1999; Xiao and Jang, 2000; Pickart, 2001).
Prominent among these, and certainly most relevant here,
are the SCF ubiquitin protein ligases (for Skp1p, Cdc53p/
cullin, and F-box protein in yeast and mammals) (reviewed
by Deshaies, 1999; Xiao and Jang, 2000). SCF complexes
are multimeric enzymes of at least four subunits built around
a member of the cullin family of proteins (Mathias et al.,
1996). In conjunction with a second subunit, RBX1 (also
known as ROC1 or HRT1), the cullin catalyzes polyubiquitin
chain formation by interacting with the E2 enzyme (Seol et
al., 1999). The cullin also binds to a member of the SKP1
protein family, which, in turn, binds to an F-box protein, so
called because it contains an N-terminal F-box motif required for its interaction with SKP1 (Skowyra et al., 1997;
Kishi and Yamao, 1998; Patton et al., 1998). The C terminus
of the F-box protein consists of any one of a number of protein–protein interaction domains that bind the relevant target protein, making it accessible to the E2-SCF complex
(Kishi and Yamao, 1998; Patton et al., 1998).
Our knowledge of the SCF system is derived mainly from
extensive work in yeast and mammals (Patton et al., 1998;
Deshaies, 1999; Gray and Estelle, 2000; Jackson et al.,
2000). There is an ever-growing number of examples of SCF
activity in signal transduction, from the derepression of Glc
transporter genes in response to Glc in Saccharomyces cerevisiae (Bernard and Andre, 2001) to the control of NF- B
activity in response to infection in mice (reviewed by Karin
S82
The Plant Cell
Figure 1. Auxin Regulates the Ubiquitination of Target Proteins, Marking Them for Degradation by the 26S Proteasome.
Key components of this pathway are shown. Target proteins are recruited to the E3 SCF ligase by the F-box protein. Auxin-regulated modification of targets, which include the Aux/IAA proteins, is likely to be required before recognition by the F-box protein. Ubiquitin (Ub) is activated (via
an E1 enzyme) and conjugated to the target (via E2–E3 interaction), a process that is reiterated to form a polyubiquitin chain. The ubiquitination
activity of the SCF is enhanced by modification of the cullin subunit with the ubiquitin-like protein RUB1. RUB1 is activated by AXR1/ECR1, an
E1-like enzyme. The COP9 signalosome regulates the deconjugation of RUB1 (dotted arrow) and possibly other processes relating to the efficient degradation of SCF targets.
and Ben-Neriah, 2000). This work has established a paradigm for the involvement of the SCF in the regulation of
gene expression that is as relevant to auxin signaling in
plants as to Glc and amino acid signaling in budding yeast.
SCF-MEDIATED UBIQUITINATION MEDIATES THE
AUXIN RESPONSE
The crucial evidence for the involvement of SCF-mediated
protein degradation in auxin signaling came through the
characterization of the tir1 mutants of Arabidopsis (Table 1)
(Ruegger et al., 1998). Although the tir1 alleles were identified in a screen for resistance to inhibitors of auxin transport, the mutants were more markedly resistant to inhibitory
concentrations of auxin, suggesting a defect in auxin response rather than transport (Ruegger et al., 1998). TIR1
was found to encode an F-box protein (Ruegger et al., 1998)
and subsequently was shown to interact with either of the
Arabidopsis Skp1-like proteins, ASK1 or ASK2, and the cullin AtCUL1 to form the functional SCF TIR1 (Gray et al., 1999,
2001). The idea that regulated protein degradation via
SCFTIR1 is required for proper auxin signaling was strengthened by the analysis of mutation in ASK1 (Table 1). ask1-1
shows reduced auxin response, being slightly more auxin
resistant than tir1-1 (Gray et al., 1999). This may reflect the
fact that TIR1 has several close homologs in Arabidopsis,
indicating the potential for redundancy in TIR1 function
(Gray et al., 1999). The C-terminal domain of TIR1 and its
homologs consists of Leu-rich repeats, which presumably
are involved in target selection (Ruegger et al., 1998; Hsiung
et al., 2001). What, then, are the targets for SCF TIR1? In the
search for candidates, the Aux/IAA family of transcription
factors are likely suspects.
REGULATED Aux/IAA STABILITY IS CRITICAL FOR
AUXIN SIGNALING
The Aux/IAAs are a family of extremely short-lived nuclear
proteins (reviewed by Abel and Theologis, 1996). Aux/IAA
genes are induced rapidly by auxin and are found throughout the higher plants. Their importance in auxin signal transduction is manifest in the severe auxin-related phenotypes
arising from semidominant mutations in several of these
genes, including AXR2/IAA7, AXR3/IAA17, SHY2/IAA3,
BODENLOS/IAA12, SLR1/IAA14, MSG2/IAA19, and IAA28
(Table 1) (Rouse et al., 1998; Tian and Reed, 1999; Nagpal
et al., 2000; Rogg et al., 2001; reviewed by Reed, 2001).
Aux/IAA proteins share a typical four-domain structure (Fig-
Ubiquitination and Auxin Signaling
ure 2), and what is striking about these semidominant mutations is that they occur only within the highly conserved
domain II, centered on a core GWPPV motif. The spectacular
auxin-related phenotypic effects of these single–amino acid
substitutions illustrate the critical importance of domain II to
Aux/IAA function (Rouse et al., 1998; Tian and Reed, 1999;
Nagpal et al., 2000; Rogg et al., 2001). Domain II is the site of
the destabilization signal that confers instability on Aux/IAA
members; measured half-lives range from as little as 6 min to
80 min (Abel et al., 1994; Ouellet et al., 2001).
Translational fusions of Aux/IAA N-terminal regions containing domain II with reporter proteins such as -glucuronidase (GUS) or luciferase have shown that this lability can be
conferred on otherwise stable proteins (Worley et al., 2000;
Gray et al., 2001). The transferable destabilization signal
within Aux/IAAs has been finely mapped to a 13–amino acid
region (or so-called degron) around the core GWPPV residues in domain II. Although this 13–amino acid degron
fused to luciferase excludes an essential bipartite nuclear
localization signal spanning domain II, the use of a heterologous nuclear localization signal has shown that in the correct subcellular context, these 13 amino acids are sufficient
S83
to confer instability (Ramos et al., 2001). Further direct and
indirect measurements of Aux/IAA stability have shown that
the phenotypic consequences of mutations in domain II may
be the result of stabilization of the respective IAA proteins
without affecting their nuclear localization (Worley et al.,
2000; Gray et al., 2001; Ouellet et al., 2001). For example,
the axr3-1 mutation, a Pro-to-Leu substitution in domain II,
causes an almost sevenfold increase in protein half-life
(Ouellet et al., 2001). Among many auxin-related phenotypes (Table 1), this mutation causes increased apical dominance, adventitious rooting, and complete agravitropism in
roots (Leyser et al., 1996). Collectively, these data stress the
importance of Aux/IAAs and their instability to auxin response. So, to reverse an earlier question, what are the regulators of Aux/IAA stability?
Aux/IAA PROTEINS ARE TARGETS OF SCF TIR1
Evidence of the involvement of SCF TIR1 in Aux/IAA degradation came from the analysis of AXR3/IAA17 reporter
Table 1. Phenotypic Summary of Auxin Signaling Mutants
Mutant/Gene
Protein
Mutant Phenotype
Reference
axr2-1/IAA7
Aux/IAA
axr3-1/IAA17
axr3-3
Aux/IAA
Timpte et al., 1994;
Wilson et al., 1990;
Nagpal et al., 2000
Leyser et al., 1996;
Rouse et al., 1998
bdl/IAA12
Aux/IAA
Reduced stature, short hypocotyl and expanded leaves in the dark,
wavy leaves, agravitropic roots and shoots, reduced adventitious
rooting, no root hairs
Reduced stature, increased shoot apical dominance, short hypocotyl
and expanded leaves in the dark, curled leaves, agravitropic roots,
increased adventitious rooting, no root hairs
Reduced stature, reduced shoot apical dominance, curled leaves,
no embryonic root
Reduced shoot apical dominance, reduced lateral rooting
Agravitropic and aphototropic hypocotyl, reduced lateral rooting
Reduced stature, short hypocotyl and expanded leaves in the dark,
curled leaves, long root hairs
Kim et al., 1996;
Reed et al., 1998;
Tian and Reed, 1999;
Knox, K., Kepinski, S.,
Leyser, O., unpublished data
Reed, 2001
Berleth and Jürgens, 1993;
Hardtke and Berleth, 1998;
Mattsson et al., 1999
Harper et al., 2000
iaa28-1/IAA28 Aux/IAA
msg2-1/IAA19 Aux/IAA
msg2-2
msg2-3
msg2-4
shy2-2/IAA3
Aux/IAA
slr1-1/IAA14
mp/ARF5
Aux/IAA
ARF
Agravitropic hypocotyl and root, no lateral roots, few root hairs
Defects in root meristem initiation in the early embryo, reduced auxin
transport
nph4/ARF7
ARF
axr1-12/AXR1
axr1-3
E1 RUB1activating
enzyme
F-box protein
Aphototropic hypocotyl with reduced gravitropic response, reduced
auxin-inducible gene expression
Auxin resistant, reduced stature, reduced shoot apical dominance,
crinkled leaves, abnormal flowers, reduced lateral rooting, reduced
auxin-inducible gene expression
Auxin resistant, reduced auxin-induced hypocotyl elongation,
reduced lateral rooting
Auxin resistant, abnormal flowers, reduced lateral rooting
tir1-1/TIR1
tir1-3
ask1-1/ASK1
SKP protein
Hamann et al., 1999
Rogg et al., 2001
Reed, 2001
Estelle and Sommerville, 1987;
Lincoln et al., 1990;
Timpte et al., 1995
Ruegger et al., 1998
Gray et al., 1999;
Zhao et al., 2001
S84
The Plant Cell
used in an identical pulldown assay, the interaction with
SCFTIR1 was abolished or reduced severely (Gray et al.,
2001). The latter data are in complete accord with the measured effect of the mutations on the stability of the axr2-1
and axr3-1 proteins in planta.
AUXIN FURTHER DESTABILIZES Aux/IAA PROTEINS
Figure 2. Protein Structures of Aux/IAAs and ARFs.
Aux/IAAs and ARFs share homology in their C-terminal domains III
and IV, which mediate homodimerization and heterodimerization.
Domain III contains a motif that is important for dimerization.
Domain II of Aux/IAAs is required for the auxin-regulated destabilization of the protein. The 13 amino acids that are sufficient to confer
instability are shown, and those conserved across the Aux/IAA family are highlighted. ARFs have an N-terminal DNA binding domain
(DBD) that binds to TGTCNC-type AuxREs of auxin-regulated
genes. The amino acid composition of the middle region (MR) of
ARFs affects their ability to activate transcription.
fusion proteins in the tir1 mutant background (Gray et al.,
2001). This fusion protein consists of N-terminal domains I
and II of AXR3, including the bipartite nuclear localization
signal, fused translationally to GUS and expressed under
the control of a heat-inducible promoter in transgenic Arabidopsis. After heat induction in a wild-type background, the
fusion protein accumulates to relatively low levels and is depleted rapidly over time compared with controls. However,
the stability of the same fusion protein was increased significantly when the construct was crossed into the tir1
background, indicating that TIR1 was required for the destabilization of AXR3 in planta (Gray et al., 2001). That TIR1
activity ultimately might result in AXR3 degradation in the
26S proteasome was supported by the finding that specific
proteasome inhibitors stabilize AXR2-, AXR3-, and other
Aux/IAA-reporter fusion proteins (Gray et al., 2001; Ramos
et al., 2001).
The final step in defining a mechanistic link between Aux/
IAA proteins and the SCFTIR1 ubiquitin ligase complex came
with the direct demonstration of the interaction of AXR2 and
AXR3 with SCFTIR1 in plant extracts. The use of glutathione
S-transferase (GST)–tagged AXR2 or AXR3 in pulldown
assays with extracts from plants expressing a c-myc epitopetagged TIR1 yielded the entire SCF TIR1 complex, as identified by immunoblotting with c-myc, AtCUL1, and ASK2 antibodies (Gray et al., 2001). Further proof that this represents
a biologically relevant interaction is indicated by the fact that
when mutant GST–axr2-1 or GST–axr3-1 proteins were
Genetic data have established the importance of regulating
the stability of Aux/IAA proteins, suggesting that the plant
might respond to auxin by regulating the turnover of Aux/
IAAs. Although previous analysis had suggested that this
was not the case (Abel et al., 1994), recent work examining
the stability of Aux/IAA-reporter fusion proteins expressed
from non-auxin-responsive promoters and using different
time frames has reached different conclusions. These studies were able to uncouple any auxin-induced Aux/IAA
protein destabilization from auxin-induced Aux/IAA gene
expression to reveal a significant increase in the degradation of the Aux/IAA-reporter proteins in response to exogenously applied auxin (Gray et al., 2001; Zenser et al., 2001).
This effect of auxin is dose dependent and extremely rapid,
causing a considerable decrease in protein levels within 5
min (Gray et al., 2001; Zenser et al., 2001). In fact, it is possible that auxin might be absolutely required for Aux/IAA degradation, and if cells could be rid of basal levels of auxin,
Aux/IAA proteins might be rendered extremely stable.
Perfectly consistent with the in planta measures of Aux/
IAA stability, the in vitro pulldown assay with GST-AXR2 and
GST-AXR3 showed an auxin-induced increase in the recovery of SCFTIR1, which also occurred in a dose-dependent
manner (Gray et al., 2001). Indeed, the dose-response
curves for the reduction in Aux/IAA stability and the increase
in Aux/IAA–SCFTIR1 interaction are remarkably similar. Given
the highly conserved nature of domain II across the Aux/
IAAs, it is likely that many, if not all, members of the family
will be susceptible to auxin-induced destabilization to some
extent. Together, these observations convincingly support
the idea that auxin stimulates the rapid degradation of Aux/
IAAs by increasing their interaction with SCF TIR1 in some way.
REGULATION OF THE SCFTIR1–Aux/IAA INTERACTION
BY AUXIN
The mechanism by which auxin influences SCF TIR1-mediated Aux/IAA turnover is not understood. However, clues to
what might be happening may be gleaned from the many
characterized examples of SCF-mediated signaling pathways in yeast and mammals. SCF-mediated ubiquitination
usually is preceded by substrate modification. These changes
turn stable proteins into susceptible targets of the SCF,
and by far the most common modification is phosphorylation
Ubiquitination and Auxin Signaling
(Deshaies, 1999). A good example is that of NF- B activation in mammals. NF-B normally is sequestered in the cytoplasm, because its nuclear localization signal is masked
by the IB family of inhibitors. In response to inflammation
and other stress stimuli, IB is phosphorylated quickly,
ubiquitinated by SCF E3RS IB/-TrCP, and ultimately degraded, freeing NF-B to translocate to the nucleus to regulate gene expression (Karin and Ben-Neriah, 2000). Similarly,
auxin might increase the affinity of Aux/IAAs for SCF TIR1 by
stimulating their phosphorylation.
There is certainly an increasing body of biochemical and
genetic evidence supporting a role for kinases in auxin signaling. A recent interesting example is the identification
of an auxin-stimulated mitogen-activated protein kinase
(MAPK) in Arabidopsis roots (Mockaitis and Howell, 2000).
Because the auxin induction of the MAPK activity occurs
over a time frame very similar to that of the auxin-induced
destabilization of AXR3, these activities might be related.
Furthermore, specific inhibitors of MAPKs abolish expression from the auxin-responsive BA3 promoter in the root
elongation zone (Mockaitis and Howell, 2000). Although, in
other systems, MAPKs tend not to be involved in phosphorylation before ubiquitination, these data indicate that MAPK
activity can impinge on auxin signaling in some way. Another particularly relevant study has demonstrated that the
N-terminal half of some Aux/IAA proteins is phosphorylated
by phytochrome A in vitro (Colon-Carmona et al., 2000).
Stabilizing gain-of-function mutations in several Aux/IAAs
causes photomorphogenic development in the dark (Tian
and Reed, 1999; Nagpal et al., 2000). The simple and perhaps naïve prediction would be that light-activated, phytochrome-mediated phosphorylation would contribute to a
stabilizing rather than a destabilizing effect on Aux/IAA proteins. Although this is entirely possible, it is more likely that
the phosphorylation(s) has other effects on Aux/IAA activity
(see below). A third auxin-related kinase activity also has
been described. Mutations in the PINOID Ser/Thr kinase
cause a range of defects in auxin response (Christensen et
al., 2000). Although the involvement of kinases in auxin signal transduction is not unexpected, their potential role in
regulating the stability of Aux/IAAs is far from certain. This is
because the 13–amino acid degron of domain II that is sufficient to confer auxin-inducible destabilization on a reporter
protein does not contain any essential phosphorylation sites
(Ramos et al., 2001). This means that the regulation of Aux/
IAA stability is unorthodox, and if phosphorylation is a prerequisite of interaction with SCFTIR1, it must involve some
kind of adaptor protein.
Examples of non-phosphorylation-dependent regulation
of substrate–SCF interactions are beginning to emerge. One
such case concerns the degradation of the human transcription factor, hypoxia-inducible factor (HIF ), by an SCFrelated E3 in which substrate selection is mediated by the
von Hippel–Lindau tumor-suppressor protein (VHL) (reviewed
by Jackson et al., 2000). HIF is required for the activation
of a variety of responses to hypoxia that are repressed,
S85
when the oxygen supply is adequate, by the rapid destruction of HIF via VHL-mediated ubiquitination. The interaction of HIF with VHL was shown recently to be regulated
through the hydroxylation of a HIF Pro residue, a reaction
that requires molecular oxygen (Ivan et al., 2001; Jaakkola
et al., 2001). This modification of Pro may be relevant to the
regulation of Aux/IAA–SCFTIR1 interaction, because the most
striking feature of the consensus domain II degron is two
highly conserved tandem Pro residues. These residues
clearly are required for Aux/IAA instability, because their
substitution is the most common cause of the dominant
Aux/IAA-stabilizing mutations found in such alleles as axr3-1
and axr2-1 (Rouse et al., 1998; Tian and Reed, 1999; Nagpal
et al., 2000; Rogg et al., 2001). The HIF example of a divergence from the phosphorylation norm may tempt us to think
of other novel modes of regulating the Aux/IAA–SCF TIR1 interaction. Perhaps the most exciting possibility is the simplest one, that auxin itself can be bound or conjugated
within the degron, rendering the substrate susceptible to
degradation by SCFTIR1. This may seem far-fetched, but it is
a tantalizing prospect. Plant proteins can be modified in this
way (Napier, 1995; Walz et al., 2002), and the example of
HIF regulation shows that the signal transduction chain
can be very short, so that the environmental conditions limiting substrate modification and stability are the same conditions that demand a response.
Although without precedent elsewhere, a final possibility
is that the interaction between Aux/IAAs and SCF TIR1 increases in response to auxin because of modification (perhaps phosphorylation) of the TIR1 protein.
REGULATION OF SCFTIR1 ACTIVITY
Another point at which the regulation of Aux/IAA ubiquitination could conceivably take place is at the level of SCF
abundance, assembly, or activity. This suggestion comes
from studies on SCFGrr1 of yeast, which is involved in cell cycle progression and Glc and amino acid signaling (reviewed
by Johnston, 1999). The F-box protein Grr1 is structurally
similar to TIR1, both containing C-terminal Leu-rich repeats.
In yeast two-hybrid studies, it has been shown that the interaction between Grr1 and Skp1, part of the process of
SCFGrr1 assembly, is enhanced significantly in response to
Glc (Li and Johnston, 1997). Although this would seem to
set a precedent for SCF assembly in response to environmental stimuli that might be extended to SCF TIR1, recent
data have suggested other possible explanations. It has
since been shown that F-box proteins are inherently unstable (Galan and Peter, 1999; Mathias et al., 1999). This
instability has been shown to be the result of the autoubiquitination of the F-box protein while assembled in an SCF
(Galan and Peter, 1999), and it seems that SCF substrate
binding may confer protection on the F-box protein (Deshaies,
1999; Galan and Peter, 1999). Thus, the apparent increased
S86
The Plant Cell
interaction of Grr1 and Skp1 in response to Glc may reflect
increased Grr1 stability as a result of a Glc-induced increase
in the susceptibility of SCFGrr1 targets. Similarly, the stability
and abundance of TIR1 might be important for normal auxin
sensitivity, because overexpression of TIR1 in transgenic
plants results in increased auxin response (Gray et al., 1999).
These phenotypes are dependent on additional TIR1 participating in an SCF complex, because overexpression of a
TIR1 variant in which the F-box had been mutated does not
result in auxin response phenotypes (Gray et al., 1999). Furthermore, loss-of-function tir1 alleles appear to be semidominant, suggesting haploinsufficiency (Ruegger et al.,
1998). Therefore, auxin responses could be brought about
by changes in the abundance of TIR1. This explanation
could account for some of the difference in auxin responsiveness observed between tissues, because TIR1 is not expressed uniformly throughout the plant (Gray et al., 1999).
Another important event affecting the activity, rather than
the assembly, of SCFTIR1 has been shown to be crucial for
normal auxin signaling. It involves the modification of the
cullin subunit of the SCF by its conjugation with a ubiquitinlike protein called RUB1 (NEDD8 in humans and fission
yeast) that is important for optimal SCF function (del Pozo et
al., 1998). This modification with RUB1 (rubinylation) requires
AXR1, one of the first mutationally defined auxin response
genes. The axr1 mutants exhibit pleiotropic auxin-related
phenotypes indicative of wide-ranging auxin resistance,
which is more severe than that displayed by the tir1 alleles
(Table 1) (Lincoln et al., 1990; Timpte et al., 1995; Gray et al.,
1998; Ruegger et al., 1998; Stirnberg et al., 1999). Importantly, Aux/IAA-reporter protein fusions are stabilized in an
axr1 background (Gray et al., 2001), and axr1 tir1 double
mutants have a synergistic phenotype, indicating the AXR1
and TIR1 act in an overlapping pathway to destabilize Aux/
IAA proteins (Ruegger et al., 1998).
AXR1 encodes an enzymatic subunit that dimerizes with a
protein called ECR1 to form a RUB1-activating enzyme that
is structurally and functionally homologous with the E1 ubiquitin-activating enzyme (Leyser et al., 1993; del Pozo and
Estelle, 1999; del Pozo et al., 2002). Rubinylation involves
the AXR1/ECR1 dimer and an E2-like protein, RCE1. Rubinylation proceeds in a manner analogous to the enzyme
cascade that activates and conjugates ubiquitin to SCF targets, except that rubinylation does not seem to require an
E3 ligase (del Pozo and Estelle, 1999). Thus, axr1 alleles
show reduced conjugation of RUB1 to the AtCUL1 subunit
of SCFTIR1, and this is the molecular basis of the stabilization
of Aux/IAAs and auxin resistance in these mutants (del Pozo
and Estelle, 1999; Gray et al., 2001). The small amount of
RUB1 conjugation that does occur in axr1 mutants is likely
to be through the activity of another Arabidopsis gene,
AXL1, which is closely homologous with AXR1 (del Pozo et
al., 2002). Most organisms that support the modification of
cullins with ubiquitin-like proteins do so with only one or two
RUB1/NEDD8-activating enzymes (Deshaies, 1999); therefore, it seems likely that in addition to being essential for
normal auxin response, AXR1-mediated rubinylation will
have a role in the degradation of diverse substrates in numerous other signaling pathways.
Recent results suggest that the removal of RUB1 from
SCFTIR1 also is important for auxin signaling (Schwechheimer
et al., 2001) and that this activity might be mediated by the
COP9 signalosome (CSN) (Lyapina et al., 2001). The COP9
signalosome is a multiprotein complex that seems to be the
enzymatic equivalent of a Swiss army knife, with attributed
activities including phosphorylation, nucleocytoplasmic partitioning, interaction with the 26S proteasome, and now
derubinylation/deneddylation (Kwok et al., 1999; Tomoda et
al., 1999; Bech-Otschir et al., 2001; Schwechheimer et al.,
2001; reviewed by Wei et al., 1998; Wei and Deng, 1999;
Schwechheimer and Deng, 2001). Indeed, in the past 2
years of published research, the COP9 complex has gone
from being only tangentially involved to being seemingly indispensable in the function of several E3-mediated degradation systems.
The COP9 signalosome was originally identified in screens
for light signaling mutants in Arabidopsis (Chamovitz et
al., 1996). Mutation in any one of its eight subunits leads to
the loss of the entire complex and results in photomorphogenesis regardless of the presence or absence of light signals. In addition to this phenotype, null mutations in the
COP9 subunits also result in seedling lethality. Using an antisense approach directed against the CSN5/JAB1 subunit,
it has been possible to impose a less severe loss of COP9
function so that plants develop to adulthood despite reduced COP9 levels (Schwechheimer et al., 2001). Unexpectedly, the adult phenotype of these plants is similar to
the axr1 mutant phenotype and includes auxin-resistant root
elongation, reduced root branching, and increased shoot
branching (Schwechheimer et al., 2001). When the antisense CSN5 construct was crossed into the axr1-3 background, a synergistic phenotype was observed, suggesting
that the COP9 complex and AXR1 act in overlapping pathways (Schwechheimer et al., 2001).
Consistent with this idea, the stability of an Aux/IAA-luciferase fusion protein was increased in the CSN5 antisense background (Schwechheimer et al., 2001). Furthermore, the COP9
signalosome was found to coimmunoprecipitate with
SCFTIR1, and AtCUL1 was shown to interact with the CSN2
subunit in a yeast two-hybrid assay (Schwechheimer et al.,
2001). Despite the similar phenotypes resulting from AXR1
loss of function and COP9 partial loss of function, paradoxically, COP9 signalosome mutants accumulate AtCUL1RUB1 conjugates, unlike axr1 mutants (Schwechheimer et
al., 2001). This finding suggests that the removal of RUB1
from AtCUL1 and its addition is required for SCF TIR1-mediated ubiquitination and subsequent degradation by the 26S
proteasome. Interestingly, each of the eight subunits of the
COP9 signalosome is related to one of the eight subunits of
the lid of the 26S proteasome, part of the 19S particle that
feeds substrates to the proteolytic core of the complex. Perhaps, then, COP9 recognizes RUB1/NEDD8 in the same
Ubiquitination and Auxin Signaling
way that the lid of the 26S proteasome recognizes ubiquitin.
RUB1/NEDD8 deconjugation by COP9 may be analogous to
the deubiquitination activity of subunits of the 19S regulatory particle. Alternatively, COP9 may simply recognize the
ubiquitinated target protein and possess an associated activity against RUB1/NEDD8.
So what is the function of RUB1/NEDD8 conjugation to
cullin and the significance of its subsequent removal? It has
been shown that NEDD8 modification of cullins promotes SCF
ubiquitin ligase activity (Furukawa et al., 2000; Morimoto et
al., 2000; Podust et al., 2000; Wu et al., 2000; Lyapina et al.,
2001). Furthermore, it was demonstrated recently that NEDD8
is required for the efficient interaction of E2 ubiquitin with the
SCF complex (Kawakami et al., 2001). As mentioned above,
polyubiquitin chains of more than four ubiquitins are required for efficient proteolysis. Perhaps cycles of RUB1/NEDD8
conjugation and deconjugation are required to sequentially
attract E2 ubiquitin, thereby promoting polyubiquitin chain
formation. Alternatively, RUB1/NEDD8 deconjugation could
be required to release oligoubiquitinated substrates (two to
three ubiquitins) from the E2-SCF complex so that they can
be polyubiquitinated efficiently by E4 multiubiquitin-promoting enzymes.
Although these are formal possibilities, the effects of CSN
deficiency in fission yeast suggest that a reduction in the
ubiquitin ligase activity of SCFTIR1 may not explain the increased stability of the Aux/IAA-reporter fusion protein
and the auxin-related phenotypes in COP9-deficient backgrounds. Similar to the accumulation of RUB1-AtCUL1 conjugates in COP9-deficient Arabidopsis, CSN-deficient yeast
accumulate NEDD8-modified cullin, but the effect is to increase rather than decrease SCF ubiquitin ligase activity
(Lyapina et al., 2001). Because these data suggest that
COP9-mediated RUB1/NEDD8 deconjugation affects a step
between the initial ubiquitination of the target and its degradation, another possibility is that COP9 affects the delivery
of ubiquitinated substrates to the 26S proteasome. Indeed,
it has been shown that the export of the COP9 subunit
CSN5 from the nucleus to the cytoplasm is associated with
the destruction of p27 kip1, a mammalian cyclin-dependent
kinase inhibitor. p27kip1 is phosphorylated and ubiquitinated
in the nucleus, but its degradation is dependent on it being
escorted to the cytoplasm by CSN5/JAB1 (Tomoda et al.,
1999; Podust et al., 2000), where it is likely surrendered to
cytoplasmic proteasomes. Thus, the stabilization of Aux/IAA
proteins in a CSN5-deficient background may be attributable to their poor delivery to the 26S proteasome rather than
to their reduced polyubiquitination. Taking the proteasome delivery idea one step further, Schwechheimer and Deng (2001)
recently proposed that COP9 could pass ubiquitinated substrates to the proteolytic core of the proteasome directly,
supplanting its bona fide lid and thereby ensuring the efficient delivery of target proteins.
Studies in CSN5-deficient fission yeast have suggested
that if COP9 does have a role in the nucleocytoplasmic partitioning of ubiquitinated substrates, this is not accompa-
S87
nied by any large change in the subcellular distribution of
cullins (Lyapina et al., 2001). So, if ubiquitinated substrates
are moved in this way but are not accompanied by an intact
SCF, what would be the function of the RUB1/NEDD8 deconjugation of cullins? There are two possibilities. First, the
RUB1/NEDD8 deconjugation may allow the release of the
ubiquitinated substrate, possibly by prompting the disassembly of the SCF. Second, it is possible that COP9 mediates two functionally distinct events after ubiquitination:
derubinylation/deneddylation and delivery of substrates to
the proteasome. Recent data indicate that RUB1/NEDD8
modification is preceded by SCF assembly and possibly
binding of the substrate by the F-box protein (Kawakami et
al., 2001). Thus, the RUB1/NEDD8 deconjugation function
of COP9 might be entirely unrelated to its substrate delivery
function, acting to prevent the unnecessary interaction of
ubiquitin-laden E2 with cullin, which was not part of a complete and ready SCF.
The activity of the RUB1 conjugation pathway in Arabidopsis apparently is unaffected by the addition of auxin, indicating that it is not a direct route for auxin signal
transduction (del Pozo et al., 2002). Nevertheless, AXR1
clearly is crucial for normal auxin response, but is it important only in auxin response? Because it appears that all
SCFs are subject to RUB1 modification, defects other than
those relating to the auxin response might be expected, but
these are not apparent in axr1 mutants. It is possible that
axr1 mutants are so crippled by their defective auxin signaling that other phenotypes are masked. However, mutations
in the F-box protein UFO, part of the putative SCF UFO, cause
abnormal flower development that is not observed in an axr1
background (Samach et al., 1999; del Pozo et al., 2002).
This reflects either a differential sensitivity for RUB1 modification in the UFO pathway or a greater dependence on rubinylation via the AXR1 homolog AXL1 (del Pozo et al., 2002).
Although there is a differential requirement for RUB modification among SCFs in budding yeast (Lammer et al., 1998;
Liakopoulos et al., 1998), it seems to be required more generally by SCFs in other eukaryotes (del Pozo and Estelle,
1999; Furukawa et al., 2000; Osaka et al., 2000; Gray et al.,
2001). Analysis of the loss of AXL1 function should shed
light on the extent to which RUB1 modification affects the
responses mediated by diverse SCFs in Arabidopsis.
Aux/IAA PROTEIN FUNCTION
The defects in auxin signaling caused by mutations in Aux/
IAAs and ubiquitin pathway components demonstrate the
consequences of failing to regulate the stability of Aux/IAAs.
But what is the function of the strict regulation of Aux/IAA
abundance?
Aux/IAAs are nuclear proteins, and their activities as transcriptional regulators appear to be based on their ability
to form a variety of dimers. First, individual Aux/IAAs are
S88
The Plant Cell
able to both homodimerize and heterodimerize with other
members of the Aux/IAA family (Kim et al., 1997). These interactions require domains III and IV, and for some homodimerization events, domain I (Figure 2) (Kim et al., 1997;
Ouellet et al., 2001). Although the function of Aux/IAA dimerization is not clear, evidence of its importance may be inferred from the fact that intragenic revertant mutations that
suppress the axr3-1 phenotype (Table 1) compromise the
ability of the protein to form dimers (Ouellet et al., 2001). Domain III contains a predicted motif, which is characteristic of prokaryotic repressors that bind DNA as tetramers
(Abel et al., 1994; Morgan et al., 1999). Indeed, in isolation,
AXR3 domain III polypeptides were able to form dimers, trimers, and tetramers (Ouellet et al., 2001), indicating the
possibility that full-length Aux/IAA proteins also may be able
to form higher order multimers. Therefore, although the role
of the domain III motif may be only in the efficient formation of dimers/multimers, it is possible that Aux/IAAs can
bind DNA directly and perhaps modulate transcription.
Aux/IAAs INTERACT WITH AUXIN RESPONSE
FACTOR PROTEINS
Aux/IAAs can also heterodimerize with members of the
auxin response factor (ARF) family of transcription factors
(Kim et al., 1997; Ulmasov et al., 1997a, 1997b). ARFs share
considerable homology in their C termini with the Aux/IAAs,
possessing the conserved domains III and IV (Figure 2)
(Ulmasov et al., 1997a). Interaction via these ARF domains
allows dimerization with Aux/IAAs and also the formation of
homodimers and heterodimers with other ARFs (Kim et al.,
1997). Unlike Aux/IAAs, ARFs have been shown to bind
DNA directly by virtue of a large B3-type DNA binding domain toward their N termini (Ulmasov et al., 1997a, 1999b).
For the ARFs characterized to date, this binding is directed
specifically to auxin response elements (AuxREs) containing
TGTCNC motif(s) (typically TGTCTC) in various contexts that
often involve coupling elements (Ulmasov et al., 1997a;
Guilfoyle et al., 1998). Synthetic palindromic or direct repeats of these six nucleotides are sufficient to bind ARFs
and confer auxin regulation on the transcription of a reporter
gene (Ulmasov et al., 1997b, 1999a, 1999b). Hence, ARFs
appear to mediate auxin-regulated gene expression through
binding to AuxREs, and Aux/IAAs have the potential to alter
the transcription of auxin-inducible genes by interacting
with ARFs.
The number of interactions within and between Aux/IAAs
and ARFs is potentially vast. Both the Aux/IAAs and ARFs
constitute large families in Arabidopsis, with 25 Aux/IAA
members and 20 ARFs (Reed, 2001). Tissue-specific and
developmental variations in the expression of both families
suggest that some of these proteins, although capable of
dimerizing, will be separated spatially and temporally in the
growing plant (Abel et al., 1995; Hardtke and Berleth, 1998).
Nevertheless, the interactions are likely to be complex, reflecting the need for fine control of auxin induction of numerous genes.
Our understanding of the interactions of ARFs and Aux/
IAAs that regulate transcription from auxin-inducible promoters is based largely on work with carrot cell suspension
cultures, in which TGTCTC-containing AuxRE promoter–
reporter fusions were used to monitor the effects of the coexpression of various ARF- and Aux/IAA-derived genes
(Ulmasov et al., 1997b, 1999a). These experiments have
shown that ARFs can act as both repressors and activators
of transcription depending on qualitative differences in their
middle regions between the N-terminal DNA binding domain
and C-terminal domains III and IV (Figure 2) (Ulmasov et al.,
1999a). ARF1, for example, which is P/S/T rich in its middle
region, suppresses both basal and auxin-inducible expression from AuxRE-containing promoters. In contrast, expression of ARF5, ARF6, ARF7, or ARF8, which are Q rich in their
middle regions, increases both basal and auxin-inducible
expression from the AuxRE-containing promoters; on the
other hand, expression of ARF2, ARF3, ARF4, or ARF9 has
no effect. These regulatory characteristics were shown to be
independent of AuxRE binding, because essentially identical
results were obtained when the ARF DNA binding domain
was replaced with the DNA binding domain from the budding
yeast GAL4 gene and simultaneously the AuxRE–reporter
gene fusion was replaced with a GAL4 binding element–
reporter gene fusion (Ulmasov et al., 1999a). These data
suggest that the auxin inducibility of AuxRE-containing promoters depends on the middle and C-terminal regions of the
ARFs, because a Q-rich middle region with a C-terminal
Aux/IAA-like dimerization domain is sufficient to confer auxin
inducibility on a GAL4 DNA binding domain promoter element system.
When the DNA binding domain was removed from the
various ARFs and these truncated versions were introduced
into carrot protoplasts with an AuxRE-reporter construct,
truncated ARFs with Q-rich middle regions still were able to
activate transcription (Ulmasov et al., 1999a). These effects
were abolished if the dimerization domains were removed
as well. Because the localization of the truncated ARFs to
the vicinity of the AuxRE likely is required, one explanation
for these results is that ARFs can dimerize with endogenous
ARFs that occupy the AuxREs through their DNA binding
domains, and subsequently, their middle regions can regulate transcription (Ulmasov et al., 1999a). Because the overexpression of these truncated ARFs increases the basal
level of reporter gene transcription, these data further indicate that the endogenous ARF with which they might interact is bound to the TGTCTC-AuxRE in the absence of
additional auxin.
In contrast to the activating ARFs, ARF1 lost its ability to
repress transcription when it was expressed without its DNA
binding domain, suggesting that part of the repression is
through the occupation of AuxREs (Ulmasov et al., 1999a).
Domain-swapping experiments with truncated ARF1 and
Ubiquitination and Auxin Signaling
ARF7 provide further clues to the mechanism of ARF1 repression. The expression of the AFR1 middle region with
ARF7 dimerization domains resulted in slight repression
(Ulmasov et al., 1999a). This repression is likely the result of
the chimeric protein titrating out endogenous activating
components rather than possessing a repressing quality per
se, reflecting a difference in the ability to interact with
AuxRE-bound factors conferred by ARF1 and ARF7 dimerization domains.
Dimerization through their C-terminal domains clearly is
important to the activities of ARFs on auxin-inducible promoters. Because Aux/IAAs are able to dimerize with ARFs,
their potential to influence ARF-mediated transcription has
also been examined in the carrot protoplast system (Ulmasov
et al., 1997b). Four different Aux/IAAs were tested, and all
were found to repress transcription from TGTCTC-containing AuxREs. This finding is consistent with the idea that
Aux/IAA interaction with AuxRE-bound ARFs can reduce the
ability of ARFs to activate transcription in response to auxin.
An attractive hypothesis is that this occurs by Aux/IAAs
competing with ARFs for dimerization through domains III
and IV (Ulmasov et al., 1997b). However, the presence of
endogenous ARFs on the AuxREs in this system has not
been proven, and at present there is no direct evidence to
show ARF-Aux/IAA association on a promoter.
S89
inactivated repressor proteins, because transcription from
AuxRE-containing promoters can be activated not only by
auxin but also by inhibitors of protein synthesis such as cycloheximide (Koshiba et al., 1995). The possibility that these
unstable transcriptional inhibitors are Aux/IAA proteins is
supported by the observation that the dominant, stabilizing
mutations in domain II of the Aux/IAA proteins often result in
the constitutive repression of transcription from auxin-inducible AuxRE-containing promoters (Abel et al., 1995; Oono et
al., 1998; Nagpal et al., 2000; Rogg et al., 2001).
Although these observations fit the general principle of
the model, at first glance, other data do not. Aux/IAA genes
were defined originally by their auxin inducibility, and most
contain TGTCNC-type AuxREs in their promoters (reviewed
by Abel and Theologis, 1996; Guilfoyle et al., 1998). Hence,
at the same time that their protein levels are being depleted
by auxin-induced SCFTIR1-mediated destabilization, they are
being replenished by increased transcript accumulation.
The auxin-induced increases in Aux/IAA transcript levels often persist for many hours and outlast more modest increases in Aux/IAA protein levels (Abel et al., 1995; Oeller
and Theologis, 1995). This is not the pattern of message and
protein accumulation that would be expected if Aux/IAA
proteins repress their own transcription. On the contrary, if
A BASIC MODEL FOR AUXIN-INDUCED
GENE EXPRESSION
Combined with the data on the effect of auxin on Aux/IAA
abundance, these observations have led to a model of
auxin-regulated transcription from TGTCTC-containing
AuxREs (Figure 3). At basal levels of auxin, Aux/IAAs are relatively stable and dimerize with ARF proteins on AuxREs,
reducing transcription by preventing the recruitment of activating ARFs. Increasing auxin levels cause an increasing
proportion of Aux/IAAs to be degraded, allowing the formation of a greater number of ARF-ARF dimers on AuxREs,
and hence higher levels of transcription of auxin-responsive
genes. Although this model lacks details, such as the level
of multimerization of the interacting Aux/IAAs and ARFs, it
seems a reasonable place to start: a point from which the
unquestioned complexity can be built in.
How far does this model go toward explaining the existing
genetic and molecular data on auxin-induced gene expression? Mutations in two of the Q-rich ARFs, MP/ARF5 and
NPH4/ARF7 (Table 1), confer a range of auxin-related phenotypes, including reduced expression from TGTCTC-type
AuxRE-containing auxin-inducible promoters (Hardtke and
Berleth, 1998; Harper et al., 2000). This finding is consistent
with the idea that Q-rich ARFs are required to activate the
expression of auxin-inducible genes in vivo. There also is
good evidence that transcription from TGTCTC AuxRE-regulated genes usually is kept inactive by very unstable auxin-
Figure 3. Model for Aux/IAA Action.
Aux/IAAs form a variety of dimers with both ARFs and other Aux/
IAAs. The equilibrium of these dimerization events has the potential
to alter gene expression from TGTCNC-containing AuxREs in auxinresponsive promoters. Auxin regulates both the degradation of Aux/
IAA proteins and the transcription of Aux/IAA genes. Given that the
diverse patterns of degradation and expression with the large Aux/
IAA family would lead to considerable temporal variation in the relative abundance of different Aux/IAAs, the interactions are likely to be
very complex.
S90
The Plant Cell
this were the case, then Aux/IAA mRNA levels should decrease as Aux/IAA protein levels increase.
One explanation might be that the model is based on
transcriptional activation from TGTCTC-type AuxREs and
does not include the contributions of other elements that affect the auxin induction of some Aux/IAAs (Ballas et al.,
1995) and other auxin-responsive genes (Guilfoyle et al.,
1998). These other elements may act as enhancers (Ballas
et al., 1995) or may require other activating factors that have
not been identified. If other factors are involved, the regulation of their activity would seem to require the involvement
of the AXR1 pathway, because axr1 mutants are defective in
virtually every measurable aspect of auxin signaling (Table
1). Therefore, although unproven, the possibility still exists
that the motifs of Aux/IAAs and ARFs might allow direct
DNA binding. Because the important regulatory step of
TGTCTC AuxRE induction seems to be the recruitment of
activating ARFs with Q-rich middle regions (Ulmasov et al.,
1999a), unorthodox ARF binding to other sites, possibly in
cooperation with Aux/IAAs, also might bring about transcription. In any case, it is possible that other elements
could contribute to longer term induction after the very rapid
induction from TGTCTC-type AuxREs.
The Aux/IAAs are a large and diverse family; therefore, a
second possibility is that in addition to the negative effect of
some Aux/IAAs, others might indirectly have a positive effect on ARF-mediated gene expression. In the period after
an auxin pulse, the balance of dimerization events could be
biased toward allowing activating ARFs to interact on
AuxREs. For example, newly synthesized individual Aux/
IAAs that normally would block activating ARF dimers might
themselves be sequestered and titrated out by other Aux/
IAAs, which usually are at very low abundance when auxin
levels are low. Similarly, nonactivating and repressing ARFs
might be prevented from interfering with activating ARF
dimerization at the AuxRE. This possibility would require
significant temporal variation in the relative abundance of
different Aux/IAAs.
Such variation is indeed reflected in the data. There are
25 different Aux/IAAs with varying temporal and dose responses (Abel et al., 1995). For example, some Aux/IAAs are
induced very rapidly by auxin: SHY2/IAA3 message reaches
almost maximal levels within 10 min, whereas the induction of AXR2/IAA7 is much slower, taking several hours to
approach similar levels (Abel et al., 1995). Furthermore, the
induction of IAA7 and IAA8 is insensitive to cycloheximide,
suggesting that they form part of a secondary response to
the auxin stimulus. Also, the limited data on Aux/IAA turnover show large differences in the stability of Aux/IAAs, the
half-life of IAA7 being much shorter (11 min) than that of
AXR3/IAA17 (80 min) (Gray et al., 2001; Ouellet et al.,
2001). It also seems likely that there will be variation in the
auxin-induced destabilization dynamics of Aux/IAAs (Gray
et al., 2001).
Coupled with the possibility of different binding affinities
among the interacting components, the functional signifi-
cance of these differences may be to swing the balance of
dimerization events between ARFs and Aux/IAAs initially toward, and then eventually away from, promoting the interaction of activating ARFs at the AuxRE. The idea that different
Aux/IAAs do different things at different times in response to
auxin certainly is supported by the genetic data. The similar
stabilizing mutations of Aux/IAAs confer quite different and
even opposite phenotypes. For example, the axr3-1 mutant
has increased adventitious rooting, whereas the axr2-1 mutant has fewer adventitious roots than the wild type (Table 1)
(Leyser et al., 1996; Nagpal et al., 2000).
It is possible that in the period after auxin stimulation, the
activity of newly synthesized Aux/IAAs is affected by posttranslational modifications that contribute to a prolonged
response in the face of accumulating Aux/IAA protein. For
example, phosphorylation of Aux/IAAs might alter their
dimerization preferences to effect a bias toward the activation of AuxREs or even allow the direct stabilization and enhancement of activating ARF binding at the AuxRE. Indeed,
if the phosphorylating activities of the auxin-induced MAPK,
phytochrome A, or PINOID kinase act in any way through
Aux/IAAs, it might be in modulating their activity in this manner. In these scenarios, there are parallels with the previously described activation and repression of the mammalian
transcription factor NF-B. Under normal stress conditions,
the ubiquitin-mediated degradation of its inhibitors allows
NF-B to activate both the genes required to respond to
stress and the expression of its own inhibitor, IB, ending
the response (Karin and Ben-Neriah, 2000). However, in response to severe stress stimuli, such as Human immunodeficiency virus infection, for example, NF-B activation is
extended despite the accumulation of its inhibitors (DeLuca
et al., 1999). This is because a hypophosphorylated version
of another of its inhibitors, IB, no longer inhibits but protects the binding of NF-B to its DNA targets and prevents
inhibition by IB (DeLuca et al., 1999).
AUXIN PERCEPTION
Clearly, auxin stimulation sets off a momentous chain of
events in the nucleus, leading to the differential expression
of hundreds of genes. However, despite valiant efforts, the
mechanism by which the auxin signal is perceived is poorly
understood. Several lines of evidence suggest the possibility of several modes of perception. Naturally, the search for
auxin receptors has focused on proteins that are able to
bind auxin (reviewed by Venis and Napier, 1995). In recent
years, much attention has been directed toward the auxin
binding protein ABP1 (reviewed by Napier, 1995). ABP1 binds
auxin with high specificity and affinity, possessing a dissociation constant for the synthetic auxin naphthalene-1-acetic
acid of 5 108 M (Napier, 1995). Although it shows no homology with any other known receptor family, ABP1 does
seem to mediate several cellular responses to applied auxin,
Ubiquitination and Auxin Signaling
including tobacco mesophyll protoplast hyperpolarization
(Leblanc et al., 1999a, 1999b), the expansion of tobacco
and maize cells in culture (Jones et al., 1998; Chen et al.,
2001), tobacco mesophyll protoplast division (Fellner et
al., 1996), and stomatal closure (Gehring et al., 1998). It is
clear that ABP1 acts at the cell surface to mediate these responses, because the exogenous addition of anti-ABP1 antibodies, which are unable to enter the cell, can interfere
with the ability of auxin to bring about the responses. Paradoxically, the majority of ABP1 in the cell is retained in the
endoplasmic reticulum, where the pH is too high for strong
auxin binding (Tian et al., 1995; Henderson et al., 1997).
Transgenic approaches to alter ABP1 levels have resulted in
relatively modest phenotypes that in general seem to affect
the balance between cell division and cell expansion (Jones
et al., 1998; Bauly et al., 2000). Recently, however, an insertional mutant in Arabidopsis ABP1 conferring complete loss
of ABP1 function resulting in embryo lethality has demonstrated an essential role for ABP1 in plant development
(Chen et al., 2001).
Although ABP1 seems to act at the cell surface, evidence
is accumulating for the intracellular perception of auxin. This
is based largely on comparison of the effects of auxins that
differ in their transport properties into and out of cells
(Claussen et al., 1996). This idea has been strengthened by
the characterization of Arabidopsis mutants that differ in
their ability to transport auxins. For example, loss of function
in the proposed auxin uptake carrier AUX1, a member of the
amino acid permease family, results in a variety of phenotypes, including auxin-resistant root elongation and reduced
root gravitropism (Pickett et al., 1990; Bennett et al., 1996;
Marchant et al., 1999). The roots of aux1 are resistant to
membrane-impermeable auxins such as 2,4-D but respond
normally to the membrane-permeable auxin naphthylacetic
acid, showing recovery of graviresponse (Yamamoto and
Yamamoto, 1998; Marchant et al., 1999), suggesting that intracellular auxin is important for root growth inhibition. Intracellular auxin binding proteins that might mediate such
responses have been identified in rice (Kim et al., 1998). One
example is of a rice 57-kD soluble auxin binding protein that
appears to interact directly with the plasma membrane proton-pumping ATPase, suggesting a very short signaling chain
from auxin to increased proton pumping, cell wall acidification, and hence cell elongation (Kim et al., 2000, 2001).
In addition to the possible role of these auxin binding proteins, it has been proposed that proteins involved in auxin
transport might be able to perceive auxin levels by monitoring the flux of auxin through transporters (Palme and
Gälweiler, 1999). In Arabidopsis, there are four members in
the auxin influx carrier family, typified by the amino acid permease-like AUX1 (Bennett et al., 1996), and eight members
in the auxin efflux carrier family, including PIN1 and PIN2/
EIR1/AGR1/WAV6 (reviewed by Palme and Gälweiler, 1999).
It is possible that some of these auxin influx/efflux carriers
might possess a specialized receptor function in addition to,
or instead of, their transport activity. There are precedents
S91
here in the sensing of amino acids and Glc in the budding
yeast S. cerevisiae. The perception of both amino acids and
Glc has been shown to involve membrane proteins distinct
from but highly homologous with amino acid or Glc transporters. For example, the amino acid permease-like sensor
Ssy1 is structurally similar to amino acid transporters except that it contains a large N-terminal cytosolic tail that
is essential for amino acid sensing (Didion et al., 1998;
Iraqui et al., 1999). Similarly, the Glc transporter homologs Snf3 and Rgt2 are distinguished from normal
transporters by their long C-terminal cytosolic tails, which
are required for Glc perception (Özcan et al., 1998). Interestingly, the downstream signaling pathways of Ssy1 and
Snf3/Rgt2 both require the involvement of SCF Grr1, the E3
ligase containing the F-box protein Grr1 that shows considerable homology with TIR1 (Li and Johnston, 1997;
Ruegger et al., 1998; Bernard and Andre, 2001). Given the
similarity of IAA to Trp, an attractive hypothesis is that
auxin signaling may have evolved from an amino acid signaling pathway.
CONCLUSIONS
Undoubtedly, there is much left to learn about the plant’s response to auxin. Although the model for auxin-induced
gene expression provides a framework that allows the activities of SCFTIR1 and other SCFs, Aux/IAAs, and ARFs to be
conceptualized, more details are required. Specifically, we
need to understand the events that link auxin to changes in
the stability of Aux/IAAs. We also need a better characterization of the consequences of the resulting fluctuations in
Aux/IAA abundance for the balance of dimerization events
between Aux/IAAs and ARFs. This will require an appreciation of the extent of functional diversity/redundancy among
members of both families. It is relatively straightforward to
compare spatial and temporal patterns of ARF and Aux/IAA
induction and patterns of Aux/IAA instability. Addressing the
extent, if any, of the differential binding preferences among
the ARFs and Aux/IAAs in vivo is much more difficult. Largescale ARF and Aux/IAA knockout programs and transcriptomic analyses are under way at present. These approaches
should help us determine the activities of ARFs and Aux/
IAAs that lead to the differential expression of hundreds of
genes and coordinated developmental changes in response
to auxin.
ACKNOWLEDGMENTS
We thank Jerry Cohen and Jane Murfett for helpful discussions and
Rebecca Garrod for critical reading of the manuscript.
Received October 10, 2001; accepted February 5, 2002.
S92
The Plant Cell
REFERENCES
Abel, S., and Theologis, A. (1996). Early genes and auxin action.
Plant Physiol. 111, 9–17.
Abel, S., Oeller, P.W., and Theologis, A. (1994). Early auxininduced genes encode short-lived nuclear proteins. Proc. Natl.
Acad. Sci. USA 91, 326–330.
Abel, S., Nguyen, M.D., and Theologis, A. (1995). The Ps-IAA4/5like family of early auxin-inducible messenger-RNAs in Arabidopsis thaliana. J. Mol. Biol. 251, 533–549.
Azevedo, C., Santos-Rosa, M.J., and Shirasu, K. (2001). The
U-box protein family in plants. Trends Plant Sci. 6, 354–358.
Ballas, N., Wong, L.-M., Ke, M., and Theologis, A. (1995). Two
auxin-responsive domains interact positively to induce expression
of the early indoleacetic acid-inducible gene PS-IAA4/5. Proc.
Natl. Acad. Sci. USA 92, 3483–3487.
Bauly, J.M., Sealy, I.M., Macdonald, H., Brearley, J., Droge, S.,
Hillmer, S., Robinson, D.G., Venis, M.A., Blatt, M.R., Lazarus,
C.M., and Napier, R.M. (2000). Overexpression of auxin-binding
protein enhances the sensitivity of guard cells to auxin. Plant
Physiol. 124, 1229–1238.
Bech-Otschir, D., Kraft, R., Huang, X., Henklein, P., Kapelari, B.,
Pollmann, C., and Dubiel, W. (2001). COP9 signalosome-specific
phosphorylation targets p53 to degradation by the ubiquitin system. EMBO J. 20, 1630–1639.
and Molecular Biology. (Dordrecht, The Netherlands: Kluwer Academic Publishers).
del Pozo, J.C., and Estelle, M. (1999). The Arabidopsis cullin
AtCUL1 is modified by the ubiquitin-related protein RUB1. Proc.
Natl. Acad. Sci. USA 96, 15342–15347.
del Pozo, J.C., Timpte, C., Tan, S., Callis, J., and Estelle, M.
(1998). The ubiquitin-related protein RUB1 and auxin response in
Arabidopsis. Science 280, 1760–1763.
del Pozo, J.C., Dharmasiri, S., Hellmann, H., Walker, L., Gray,
W.M., and Estelle, M. (2002). AXR1–ECR1-dependent conjugation of RUB1 to the Arabidopsis cullin AtCul1 is required for Auxin
response. Plant Cell 14, 421–433.
DeLuca, C., Petropoulos, L., Zmeureanu, D., and Hiscott, J.
(1999). Nuclear IB maintains persistent NF-B activation in HIV1-infected myeloid cells. J. Biol. Chem. 274, 13010–13016.
Deshaies, R.J. (1999). SCF and cullin/RING-H2-based ubiquitin
ligases. Annu. Rev. Cell Dev. Biol. 15, 435–467.
Didion, T., Regenberg, B., Jorgensen, M.U., Kielland-Brandt,
M.C., and Andersen, H.A. (1998). The permease homologue
Ssy1p controls the expression of amino acid and peptide transporter genes in Saccharomyces cerevisiae. Mol. Microbiol. 27,
643–650.
Estelle, M., and Sommerville, C. (1987). Auxin resistant mutants of
Arabidopsis thaliana with altered morphology. Mol. Gen. Genet.
206, 200–206.
Bennett, M.J., Marchant, A., Green, H.G., May, S.T., Ward, S.P.,
Millner, P., Walker, A., Schulz, B., and Feldmann, K. (1996).
Arabidopsis AUX1 gene: A permease-like regulator of root gravitropism. Science 273, 948–950.
Fellner, M., Ephritikhine, G., Barbier-Brygoo, H., and Guern, J.
(1996). An antibody raised to a maize auxin-binding protein has
inhibitory effects on cell division of tobacco mesophyll protoplasts. Plant Physiol. Biochem. 34, 133–138.
Berleth, T., and Jürgens, G. (1993). The role of the MONOPTEROS
gene in organizing the basal body region of the embryo. Development 118, 575–587.
Furukawa, M., Zhang, Y., McCarville, J., Ohta, T., and Xiong, Y.
(2000). The CUL1 C-terminal sequence and ROC1 are required for
efficient nuclear accumulation, NEDD8 modification, and ubiquitin
ligase activity of CUL1. Mol. Cell. Biol. 20, 8185–8197.
Berleth, T., and Sachs, T. (2001). Plant morphogenesis: Long-distance coordination and local patterning. Curr. Opin. Plant Biol. 4,
57–62.
Bernard, F., and Andre, B. (2001). Ubiquitin and the SCFGrr1 ubiquitin ligase complex are involved in the signalling pathway activated by external amino acids in Saccharomyces cerevisiae.
FEBS Lett. 496, 81–85.
Chamovitz, D.A., Wei, N., Osterlund, M.T., von Arnim, A.G.,
Staub, J.M., Matsui, M., and Deng, X.-W. (1996). The COP9
complex, a novel multisubunit nuclear regulator involved in light
control of a plant developmental switch. Cell 86, 115–121.
Chen, J.G., Ullah, H., Young, J.C., Sussman, M.R., and Jones,
A.M. (2001). ABP1 is required for organized cell elongation and
division in Arabidopsis embryogenesis. Genes Dev. 15, 902–911.
Christensen, S.K., Dagenais, N., Chory, J., and Weigel, D. (2000).
Regulation of auxin response by the protein kinase PINOID. Cell
100, 469–478.
Claussen, M., Luthen, H., and Bottger, M. (1996). Inside or outside? Localization of the receptor relevant to auxin-induced
growth. Physiol. Plant. 98, 861–867.
Colon-Carmona, A., Chen, D.L., Yeh, K.C., and Abel, S. (2000).
Aux/IAA proteins are phosphorylated by phytochrome in vitro.
Plant Physiol. 124, 1728–1738.
Davies, P.J., ed (1995). Plant Hormone Physiology, Biochemistry
Galan, J., and Peter, M. (1999). Ubiquitin-dependent degradation
of multiple F-box proteins by an autocatalytic mechanism. Proc.
Natl. Acad. Sci. USA 96, 9124–9129.
Gehring, C.A., McConchie, R.M., Venis, M.A., and Parish, R.W.
(1998). Auxin-binding-protein antibodies and peptides influence
stomatal opening and alter cytoplasmic pH. Planta 205, 581–586.
Gray, W.M., and Estelle, M. (2000). Function of the ubiquitin-proteasome pathway in auxin response. Trends Biochem. Sci. 25,
133–138.
Gray, W.M., Ostin, A., Sandberg, G., Romano, C.P., and Estelle,
M. (1998). High temperature promotes auxin-mediated hypocotyl
elongation in Arabidopsis. Proc. Natl. Acad. Sci. USA 95, 7197–
7202.
Gray, W.M., del Pozo, J.C., Walker, L., Hobbie, L., Risseeuw, E.,
Banks, T., Crosby, W., Yang, M., Ma, H., and Estelle, M. (1999).
Identification of an SCF ubiquitin-ligase complex required for
auxin response in Arabidopsis thaliana. Genes Dev. 13, 1678–
1691.
Gray, W.M., Kepinski, S., Rouse, D., Leyser, O., and Estelle, M.
(2001). Auxin regulates SCFTIR1-dependent degradation of Aux/
IAA proteins. Nature 414, 271–276.
Guilfoyle, T., Hagen, G., Ulmasov, T., and Murfett, J. (1998). How
does auxin turn on genes? Plant Physiol. 118, 341–347.
Ubiquitination and Auxin Signaling
Hamann, T., Mayer, U., and Jürgens, G. (1999). The auxin-insensitive bodenlos mutation affects primary root formation and apicalbasal patterning in the Arabidopsis embryo. Development 126,
1387–1395.
Hardtke, C.S., and Berleth, T. (1998). The Arabidopsis gene
MONOPTEROS encodes a transcription factor mediating embryo
axis formation and vascular development. EMBO J. 17, 1405–
1411.
Harper, R.M., Stowe-Evans, E.L., Luesse, D.R., Muto, H., Tatematsu,
K., Watahiki, M., Yamamoto, K., and Liscum, E. (2000). The
NPH4 locus encodes the auxin response factor ARF7, a conditional regulator of differential growth in aerial Arabidopsis tissue.
Plant Cell 12, 757–770.
Henderson, J., Bauly, J.M., Ashford, D.A., Oliver, S.C., Hawes,
C.R., Lazarus, C.M., Venis, M.A., and Napier, R.M. (1997).
Retention of maize auxin-binding protein in the endoplasmic
reticulum: Quantifying escape and the role of auxin. Planta 202,
313–323.
Hsiung, W.G., Chang, H.C., Pellequer, J.L., La Valle, R., Lanker,
S., and Wittenberg, C. (2001). F-box protein Grr1 interacts with
phosphorylated targets via the cationic surface of its leucine-rich
repeat. Mol. Cell. Biol. 21, 2506–2520.
Iraqui, I., Vissers, S., Bernard, F., De Craene, J.O., and Boles, E.
(1999). Amino acid signaling in Saccharomyces cerevisiae: A permease-like sensor of external amino acids and F-box protein
Grr1p are required for transcriptional induction of the AGP1 gene,
which encodes a broad-specificity amino acid permease. Mol.
Cell. Biol. 19, 989–1001.
Ivan, M., Kondo, K., Yang, H.F., Kim, W., Valiando, J., Ohh, M.,
Salic, A., Asara, J., Lane, W., and Kaelin, W., Jr. (2001). HIF
targeted for VHL-mediated destruction by proline hydroxylation:
Implications for O2 sensing. Science 292, 464–468.
Jaakkola, P., et al. (2001). Targeting of HIF-alpha to the von HippelLindau ubiquitylation complex by O2-regulated prolyl hydroxylation. Science 292, 468–472.
Jackson, P.K., Eldridge, A.G., Freed, E., Furstenthal, L., Hsu,
J.Y., Kaiser, B., and Reimann, J. (2000). The lore of the RINGs:
Substrate recognition and catalysis by ubiquitin ligases. Trends
Cell Biol. 10, 429–439.
Johnston, M. (1999). Feasting, fasting and fermenting. Trends
Genet. 15, 29–33.
Jones, A.M., Im, K.H., Savka, M.A., Wu, M.J., DeWitt, N.G.,
Shillito, R., and Binns, A. (1998). Auxin-dependent cell expansion mediated by overexpressed auxin-binding protein 1. Science
282, 1114–1117.
Karin, M., and Ben-Neriah, Y. (2000). Phosphorylation meets ubiquitination: The control of NF-kappa B activity. Annu. Rev. Immunol. 18, 621–663.
Kawakami, T., Chiba, T., Suzuki, T., Iwai, K., Yamanaka, K.,
Minato, N., Suzuki, H., Shimbara, N., Hidaka, Y., Osaka, F.,
Omata, M., and Tanaka, K. (2001). NEDD8 recruits E2-ubiquitin
to SCF E3 ligase. EMBO J. 20, 4003–4012.
Kim, B.C., Soh, M.S., Kang, B.J., Furuya, M., and Nam, H.G.
(1996). Two dominant photomorphogenic mutations of Arabidopsis thaliana identified as suppressor mutations of hy2. Plant J. 15,
441–456.
Kim, J., Harter, K., and Theologis, A. (1997). Protein-protein inter-
S93
actions among the Aux/IAA proteins. Proc. Natl. Acad. Sci. USA
94, 11786–11791.
Kim, Y.S., Kim, D.H., and Jung, J. (1998). Isolation of a novel auxin
receptor from soluble fractions of rice (Oryza sativa L.) shoots.
FEBS Lett. 438, 241–244.
Kim, Y.S., Kim, D., and Jung, J. (2000). Two isoforms of soluble
auxin receptor in rice (Oryza sativa L.) plants: Binding property for
auxin and interaction with plasma membrane H-ATPase. Plant
Growth Regul. 32, 143–150.
Kim, Y.S., Min, J.K., Kim, D., and Jung, J. (2001). A soluble auxinbinding protein, ABP(57): Purification with anti-bovine serum albumin antibody and characterization of its mechanistic role in the
auxin effect on plant plasma membrane H-ATPase. J. Biol.
Chem. 276, 10730–10736.
Kishi, T., and Yamao, F. (1998). An essential function of Grr1 for the
degradation of Cln2 is to act as a binding core that links Cln2 to
Skp1. J. Cell Sci. 111, 3655–3661.
Koegl, M., Hoppe, T., Schlenker, S., Ulrich, H.D., Mayer, T.U.,
and Jentsch, S. (1999). A novel ubiquitination factor, E4, is
involved in multiubiquitin chain assembly. Cell 96, 635–644.
Koshiba, T., Ballas, N., Wong, L.-M., and Theologis, A. (1995).
Transcriptional regulation of PS-IAA4/5 and PS-IAA6 early gene
expression by indoleacetic acid and protein synthesis inhibitors in
pea (Pisum sativum). J. Mol. Biol. 253, 396–413.
Kwok, S.F., Staub, J.M., and Deng, X.W. (1999). Characterization
of two subunits of Arabidopsis 19S proteasome regulatory complex and its possible interaction with the COP9. J. Mol. Biol. 285,
85–95.
Lammer, D., Mathias, N., Laplaza, J.M., Jiang, W., Liu, Y., Callis,
J., Goebl, M., and Estelle, M. (1998). Modification of yeast
Cdc53p by the ubiquitin-related protein Rub1p affects function of
the SCFCdc4 complex. Genes Dev. 12, 914–926.
Leblanc, N., David, K., Grosclaude, J., Pradier, J.M., BarbierBrygoo, H., Labiau, S., and Perrot-Rechenmann, C. (1999a). A
novel immunological approach establishes that the auxin-binding
protein, Nt-abp1, is an element involved in auxin signaling at the
plasma membrane. J. Biol. Chem. 274, 28314–28320.
Leblanc, N., Perrot-Rechenmann, C., and Barbier-Brygoo, H.
(1999b). The auxin-binding protein Nt-ERabp1 alone activates an
auxin-like transduction pathway. FEBS Lett. 449, 57–60.
Leyser, H.M.O., Lincoln, C.A., Timpte, C.S., Lammer, D., Turner,
J.C., and Estelle, M. (1993). Arabidopsis auxin-resistance gene
AXR1 encodes a protein related to ubiquitin-activating enzyme
E1. Nature 304, 161–164.
Leyser, H.M.O., Pickett, F.B., Dharmasiri, S., and Estelle, M.
(1996). Mutations in the AXR3 gene of Arabidopsis result in altered
auxin response including ectopic expression from the SAUR-AC1
promoter. Plant J. 11, 403–413.
Li, F., and Johnston, M. (1997). Grr1 of Saccharomyces cerevisiae
is connected to the ubiquitin proteolysis machinery through Skp1:
Coupling glucose sensing to gene expression and cell cycle.
EMBO J. 16, 101–110.
Liakopoulos, D., Doenges, G., Matuschewski, K., and Jentsch,
S. (1998). A novel protein modification pathway related to the
ubiquitin system. EMBO J. 17, 2208–2214.
S94
The Plant Cell
Lincoln, C., Britton, J.H., and Estelle, M. (1990). Growth and
development of the axr1 mutants of Arabidopsis. Plant Cell 2,
1071–1080.
Lyapina, S., Cope, G., Shevchenko, A., Serino, G., Tsuge, T.,
Zhou, C., Wolf, D., Wei, N., Shevchenko, A., and Deshaies, R.
(2001). Promotion of NEDD8–CUL1 conjugate cleavage by COP9
signalosome. Science 292, 1382–1385.
Marchant, A., Kargul, J., May, S.T., Muller, P., Delbarre, A., PerrotRechenmann, C., and Bennett, M.J. (1999). AUX1 regulates root
gravitropism in Arabidopsis by facilitating auxin uptake within root
apical tissues. EMBO J. 18, 2066–2073.
Mathias, N., Johnson, S., Byers, B., and Goebl, M. (1999). The
abundance of cell cycle regulatory protein Cdc4p is controlled by
interactions between its F box and Skp1p. Mol. Cell. Biol. 19,
1759–1767.
Mathias, N., Johnson, S.L., Winey, M., Adams, A.E.M., Goetsch,
L., Pringle, J.R., Byers, B., and Goebl, M.G. (1996). Cdc53p acts
in concert with cdc4p and cdc34p to control the G(1)-to-S-phase
transition and identifies a conserved family of proteins. Mol. Cell.
Biol. 16, 6634–6643.
Mattsson, J., Sung, Z.R., and Berleth, T. (1999). Responses of
plant vascular systems to auxin transport inhibition. Development
126, 2979–2991.
Mockaitis, K., and Howell, S.H. (2000). Auxin induces mitogenic
activated protein kinase (MAPK) activation in roots of Arabidopsis
seedlings. Plant J. 24, 785–796.
Morgan, K.E., Zarembinski, T.I., Theologis, A., and Abel, S.
(1999). Biochemical characterization of recombinant polypeptides
corresponding to the predicted beta alpha alpha fold in Aux/IAA
proteins. FEBS Lett. 454, 283–287.
Morimoto, M., Nishida, T., Honda, R., and Yasuda, H. (2000).
Modification of cullin-1 by ubiquitin-like protein Nedd8 enhances
the activity of SCFskp2 toward p27(kip1). Biochem. Biophys. Res.
Commun. 270, 1093–1096.
Nagpal, P., Walker, L.M., Young, J.C., Sonawala, A., Timpte, C.,
Estelle, M., and Reed, J.W. (2000). AXR2 encodes a member of
the Aux/IAA protein family. Plant Physiol. 123, 563–573.
Napier, R.M. (1995). Towards an understanding of ABP1. J. Exp.
Bot. 46, 1787–1795.
Oeller, P.W., and Theologis, A. (1995). Induction kinetics of the
nuclear proteins encoded by the early indoleacetic acid-inducible
genes Ps-IAA4/5 and Ps-IAA6 in pea (Pisum sativum L.). Plant J.
7, 37–48.
Oono, Y., Chen, Q.G., Overvoorde, P.J., Kohler, C., and
Theologis, A. (1998). age mutants of Arabidopsis exhibit altered
auxin-regulated gene expression. Plant Cell 10, 1649–1662.
Osaka, F., Saeki, M., Katayama, S., Aida, N., Toh-e, A., Kominami,
K., Toda, T., Suzuki, T., Chiba, T., Tanaka, K., and Kato, S.
(2000). Covalent modifier NEDD8 is essential for SCF ubiquitinligase in fission yeast. EMBO J. 19, 3475–3484.
Palme, K., and Gälweiler, L. (1999). PIN-pointing the molecular
basis of auxin transport. Curr. Opin. Plant Biol. 2, 375–381.
Patton, E.E., Willems, A.R., and Tyers, M. (1998). Combinatorial
control in ubiquitin-dependent proteolysis: Don’t Skp the F-box
hypothesis. Trends Genet. 14, 236–243.
Pickart, C.M. (2001). Mechanisms underlying ubiquitination. Annu.
Rev. Biochem. 70, 503–533.
Pickett, F.B., Wilson, A.K., and Estelle, M. (1990). The aux1 mutation of Arabidopsis confers both auxin and ethylene resistance.
Plant Physiol. 94, 1462–1466.
Podust, V., Brownell, J., Gladysheva, T., Luo, R.-S., Wang, C.,
Coggins, M., and Pierce, J. (2000). A Nedd8 conjugation pathway is essential for proteolytic targeting of p27Kip1 by ubiquitination. Proc. Natl. Acad. Sci. USA 97, 4579–4584.
Ramos, J.A., Zenser, N., Leyser, O., and Callis, J. (2001). Rapid
degradation of Auxin/Indoleacetic Acid proteins requires conserved amino acids of domain II and is proteasome dependent.
Plant Cell 13, 2349–2360.
Reed, J. (2001). Roles and activities of Aux/IAA proteins in Arabidopsis. Trends Plant Sci. 6, 420–425.
Reed, J.W., Elumalai, R.P., and Chory, J. (1998). Suppressors of
an Arabidopsis thaliana phyB mutation identify genes that control
light signaling and hypocotyl elongation. Genetics 148, 1295–
1310.
Rogg, L.E., Lasswell, J., and Bartel, B. (2001). A gain-of-function
mutation in IAA28 suppresses lateral root development. Plant Cell
13, 465–480.
Rouse, D., Mackay, P., Stirnberg, P., Estelle, M., and Leyser, O.
(1998). Changes in auxin response from mutations in an AUX/IAA
gene. Plant Cell 279, 1371–1373.
Ruegger, M., Dewey, E., Gray, W.M., Hobbie, L., Turner, J., and
Estelle, M. (1998). The TIR1 protein of Arabidopsis functions in
auxin response and is related to human SKP2 and yeast Grr1p.
Genes Dev. 12, 198–207.
Samach, A., Klenz, J., Kohalmi, S., Risseeuw, E., Haughn, G.,
and Crosby, W. (1999). The UNUSUAL FLORAL ORGANS gene
of Arabidopsis is an F-box protein required for normal patterning
and growth in the floral meristem. Plant J. 20, 433–445.
Schwechheimer, C., and Deng, X.W. (2001). COP9 signalosome
revisited: A novel mediator of protein degradation. Trends Cell
Biol. 11, 420–426.
Schwechheimer, C., Serino, G., Callis, J., Crosby, W.L., Lyapina,
S., Deshaies, R.J., and Deng, X.-W. (2001). Interactions of the
COP9 signalosome with the E3 ubiquitin ligase SCFTIR1 in mediating auxin response. Science 292, 1379–1382.
Seol, J.H., et al. (1999). Cdc53/cullin and the essential Hrt1 RINGH2 subunit of SCF define a ubiquitin ligase module that activates
the E2 enzyme Cdc34. Genes Dev. 13, 1614–1626.
Ouellet, F., Overvoorde, P.J., and Theologis, A. (2001). IAA17/
AXR3: Biochemical insight into an auxin mutant phenotype. Plant
Cell 13, 829–841.
Skowyra, D., Craig, K.L., Tyers, M., Elledge, S.J., and Harper,
J.W. (1997). F-box proteins are receptors that recruit phosphorylated substrates to the SCF ubiquitin-ligase complex. Cell 91,
209–219.
Özcan, S., Dover, J., and Johnston, M. (1998). Glucose sensing
and signaling by two glucose receptors in the yeast Saccharomyces cerevisiae. EMBO J. 17, 2566–2573.
Stirnberg, P., Chatfield, S.P., and Leyser, H.M.O. (1999). AXR1
acts after lateral bud formation to inhibit lateral bud growth in Arabidopsis. Plant Physiol. 121, 839–847.
Ubiquitination and Auxin Signaling
S95
Tian, H.C., Klambt, D., and Jones, A.M. (1995). Auxin-binding protein-1 does not bind auxin within the endoplasmic-reticulum
despite this being the predominant subcellular location for this
hormone-receptor. J. Biol. Chem. 270, 26962–26969.
Walz, A., Park, S., Slovin, J.P., Ludwig-Muller, J., Momonoki,
Y.S., and Cohen, J.D. (2002). A gene encoding a protein modified
by the phytohormone indoleacetic acid. Proc. Natl. Acad. Sci.
USA 99, 1718–1723.
Tian, Q., and Reed, J.W. (1999). Control of auxin-regulated root
development by the Arabidopsis thaliana SHY2/IAA3 gene. Development 126, 711–721.
Wei, N., and Deng, X.W. (1999). Making sense of the COP9 signalosome: A regulatory protein complex conserved from Arabidopsis
to human. Trends Genet. 15, 98–103.
Timpte, C., Wilson, A.K., and Estelle, M. (1994). The axr2-1 mutation of Arabidopsis thaliana is a gain-of-function mutation that disrupts an early step in auxin response. Genetics 138, 1239–1249.
Wei, N., Tsuge, T., Serino, G., Dohmae, N., Takio, K., Matsui, M.,
and Deng, X.W. (1998). The COP9 complex is conserved
between plants and mammals and is related to the 26S proteasome regulatory complex. Curr. Biol. 8, 919–922.
Timpte, C., Lincoln, C., Pickett, F.B., Turner, J., and Estelle, M.
(1995). The AXR1 and AUX1 genes of Arabidopsis function in separate auxin-response pathways. Plant J. 8, 561–569.
Tomoda, K., Kubota, Y., and Kato, J. (1999). Degradation of
cyclin-dependent-kinase inhibitor p27Kip1 is instigated by Jab1.
Nature 398, 160–165.
Ulmasov, T., Hagen, G., and Guilfoyle, T.J. (1997a). ARF1, a transcription factor that binds to auxin response elements. Science
276, 1865–1868.
Ulmasov, T., Murfett, J., Hagen, G., and Guilfoyle, T.J. (1997b).
Aux/IAA proteins repress expression of reporter genes containing
natural and highly active synthetic auxin response elements. Plant
Cell 9, 1963–1971.
Ulmasov, T., Hagen, G., and Guilfoyle, T.J. (1999a). Activation and
repression of transcription by auxin-response factors. Proc. Natl.
Acad. Sci. USA 96, 5844–5849.
Ulmasov, T., Hagen, G., and Guilfoyle, T.J. (1999b). Dimerization
and DNA binding of auxin response factors. Plant J. 19, 309–319.
Wilson, A.K., Pickett, F.B., Turner, J.C., and Estelle, M. (1990). A
dominant mutation in Arabidopsis confers resistance to auxin,
ethylene and abscisic acid. Mol. Gen. Genet. 222, 377–383.
Worley, C.K., Zenser, N., Ramos, J., Rouse, D., Leyser, O., and
Callis, J. (2000). Degradation of Aux/IAA proteins is essential for
normal auxin signalling. Plant J. 21, 553–562.
Wu, K., Chen, A., and Pan, Z.Q. (2000). Conjugation of Nedd8 to
CUL1 enhances the ability of the ROC1–CUL1 complex to promote ubiquitin polymerization. J. Biol. Chem. 275, 32317–32324.
Xiao, W.Y., and Jang, J.C. (2000). F-box proteins in Arabidopsis.
Trends Plant Sci. 5, 454–457.
Yamamoto, M., and Yamamoto, K.T. (1998). Differential effects of
1-naphthaleneacetic acid, indole-3-acetic acid and 2,4-dichlorophenoxyacetic acid on the gravitropic response of roots in an
auxin-resistant mutant of Arabidopsis, aux1. Plant Cell Physiol.
39, 660–664.
Venis, M.A., and Napier, R.M. (1995). Auxin receptors and auxin
binding proteins. Crit. Rev. Plant Sci. 14, 27–47.
Zenser, N., Ellsmore, A., Leasure, C., and Callis, J. (2001). Auxin
modulates the degradation rate of Aux/IAA proteins. Proc. Natl.
Acad. Sci. USA 98, 11795–11800.
Voges, D., Zwickl, P., and Baumeister, W. (1999). The 26S proteasome: A molecular machine designed for controlled proteolysis.
Annu. Rev. Biochem. 68, 1015–1068.
Zhao, D., Yu, Q., Chen, M., and Ma, H. (2001). The ASK1 gene regulates B function gene expression in cooperation with UFO and
LEAFY in Arabidopsis. Development 128, 2735–2746.